Composition and method comprising growth hormone and leucine

ABSTRACT

A pharmaceutical formulation comprising a growth hormone and leucine shows a very high stability against deamidation, oxidation and cleavage of peptide bonds. The stability of the product allows for the storing and shipment thereof in a lyophilized state or in the form of a dissolved or re-dissolved formulation at ambient temperature.

This application is a continuation in part of application Ser. No.08/322,838, filed Oct. 13, 1994, now abandoned, incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a stabilized pharmaceutical formulationcomprising growth hormone, to a method of making such formulation, andthe use of leucine for stabilizing a formulation of growth hormone.

BACKGROUND OF THE INVENTION

The growth hormones from man and from the common domestic animals areproteins of approximately 191 amino acids, synthesized and secreted fromthe anterior lope of the pituitary gland. Human growth hormone consistsof 191 amino acids. Growth hormone is a key hormone involved in theregulation of not only somatic growth, but also in the regulation ofmetabolism of proteins, carbohydrates and lipids. The major effect ofgrowth hormone is to promote growth. The organ systems affected bygrowth hormone include the skeleton, connective tissue, muscles, andviscera such as liver, intestine, and kidneys.

Until the development of the recombinant technology and the cloning ofthe growth hormone gene now giving rise to production of e.g. humangrowth hormone (hGH) and Met-hGH in industrial scale, human growthhormone could only be obtained by extraction from the pituitary glandsof human cadavers. The very limited supplies of growth hormonerestricted the use thereof to longitudinal growth promotion in childhoodand puberty for treatment of dwarfism, even though it has been proposedfor inter alia treatment of short stature (due to growth hormonedeficiency, normal short stature and Turner syndrome), growth hormonedeficiency in adults, infertility, treatment of bums, wound healing,dystrophy, bone knitting, osteoporosis, diffuse gastric bleeding, andpseudoarthrosis as well as for decreasing the proportion of fat inanimals to be slaughtered for human consumption.

Pharmaceutical formulations of growth hormone tend to be unstable.Degradation products such as deamidated or sulfoxylated products anddimer or polymer forms are generated--especially in solutions of growthhormone. The predominant degradation reactions of hGH are 1) deamidationby direct hydrolysis or via a cyclic succinimide intermediate to formvarious mounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH, and D-iso-asp-hGH(Y.-C. J. Wang and M. A. Hanson. Parenteral Formulations of Proteins andPeptides: Stability and Stabilizers. J. Parenteral Science andTechnology 42 (Suppl.) (1988) 53-525; M. C. Manning, K. Patel, R. T.Borchardt. Stability of Protein Pharmaceuticals. Pharmaceutical Research6 (11) (1989) 903-918; and B. A. Johnson, J. M. Shirokawa, W. S.Hancock, M. W. Spellman, L. J. Basa and D. W. Asward. J. Biol. Chem.264, 1462-71 (1989)) and 2) oxidation of the methionine residues inpositions 14 and 125 (L. C. Teh et al., J. Biol. Chem., 262, 785-794(1987); G. W. Becker et at., Biotech. Appl. Biochem., 10, 326-337(1988); R. A. Houghten et at., Arch. Biochem. Biophys., 178, 350-355(1977); R. M. Riggin et at., Anal. Biochem., 167, 199-209 (1987); P.Gellerfors et at., Acta Paediatr. Scand (suppl), 370, 93-100 (1990); M.J. Kaufman, Pharm. Res., 7 (3) 289-292 (1990)). The major degradationproduct of hGH in lyophilized state as well as in solution is deamidatedhGH. Deamidation takes place to a major extent at the Asn in position149 and to a minor extent in position 152. hGH is also rather easilyoxidized in positions 14 and 125, especially in solution (L. C. Teh etal., J. Biol. Chem., 262, 785-794 (1987); G. W. Becker et at., Biotech.Appl. Biochem., 10, 326-337 (1988); R. A. Houghten et at., Arch.Biochem. Biophys., 178, 350-355 (1977); R. M. Riggin et at., Anal.Biochem., 167, 199-209 (1987); P. Gellerfors et al., Acta. Paediatr.Scand (suppl), 370, 93-100 (1990)).

The oxidation of hGH in solution forming sulfoxides is normally due tothe oxygen dissolved in the formulation. The solubility of oxygen indistilled water is about 200 μM M. J. Kaufman, Pharm. Res., 7 (3)289-292 (1990)). As the concentration of hGH in a formulation comprising4 IU/ml is 1.3 mg/ml corresponding to 60 nM hGH, oxygen will, at normalstoring conditions, be present in an excess of about 3000 times thestoichiometric amount for oxidation of hGH. It is not feasible to try tosolve the problem by degassing of buffers before tapping and packing theformulations.

At present, it is not believed that these deamidated forms and oxidizedforms of hGH should have toxic or altered biological activity orreceptor binding properties. However, there are indications that theconformation stability of the sulfoxides is reduced as compared tonative hGH.

For the development of a stable, dissolved formulation comprising hGH,it is important to know the rate of deamidation and formation ofsulfoxides as well as means to control the reactions. The kinetics ofdegradation depend on temperature, pH and various additives or adjuvantsin the hGH formulation.

Due to its instability, growth hormone is, at present, lyophilized andstored in the lyophilized form at 4° C. until it is reconstituted foruse in order to minimize the degradation.

The lyophilized pharmaceutical formulations comprising hGH are, atpresent, reconstituted by the patient and then stored as a solutionduring the use for a period of up to 14 days at 4° C., during which somedegradation will take place. Furthermore, the process of reconstitutionof the lyophilized growth hormone tends to provide difficulties for thepatient. Therefore, it is at present preferred to reconstitute thegrowth hormone as late as possible before use and to store and ship theformulation in a lyophilized state. The links in the distributionprocess from the manufacturer to the pharmacy are equipped for handlingthe formulations at a controlled low temperature of e.g. 4° C. whichallows for a long shelf life of up to two years.

However, the extended use of pen systems for self-medication and theexpanded field of use calls for a formulation which is stable for asufficiently long time with the end user under conditions where"sufficient" cooling is not always available. Preferably, a formulationshould be stable with the end user in a lyophilized state for about onemonth and additionally for one month in a reconstituted state in a pendevice for the intended period of use of a cartridge.

Thus, there is a need for more stable formulations of growth hormonebeing stable in a lyophilized state or in solution at a relatively hightemperature for at least about one month. Such stabilization is of verygreat importance when moving the administration of the growth hormonefrom clinics to the homes of the individuals to be treated where optimalstorage may not be available as indicated above.

Furthermore, the shift in pattern of administration of growth hormone tothe use of pen devices calls for a stable dissolved formulationcomprising growth hormone in order to facilitate the handling to beperformed by the patient. A stable dissolved formulation comprisinggrowth hormone may be produced ready to use in the form of cartridgesfitting into the pen device used by the patient who may then avoid thereconstitution of the formulation and, hence, will not have to be in thepossession of a lyophilized formulation, a suitable vehicle forreconstitution as well as the necessary skill and sterile equipment forsterile reconstitution of the formulation.

For safety reasons it will also be desirable to avoid the reconstitutionof a lyophilized formulation just before the use of the formulation.

Furthermore, it would also be advantageous to avoid the lyophilizationstep in the production of growth hormone formulations. Lyophilization isa time consuming and costly process and is also often a "bottleneck" inthe production due to the limited capacity of the freeze drier. Thus,there is a need to reduce the rate of the degradation processes in orderto allow for dissolved hGH formulations to be stable during shelf lifeand during the period of use of up to one month.

Prior attempts to stabilize hGH have not fully succeeded in preventingthe formation of dimers. The problems associated with dimer formation ise.g. noted in Becker, G. W., Biotechnology and Applied Biochemistry 9,478 (1987).

International Patent Publication No. WO 89/09614 and Australian patentapplication No. 30771/89 disclose a stable pharmaceutical formulationcontaining human growth hormone, glycine, and mannitol. Such aformulation shows improved stability during normal processing andstorage in a lyophilized state as well as in the period of use after thereconstitution.

Published European patent application No. 303 746 discloses that animalgrowth hormone may be stabilized with various stabilizers to givedecreased formation of insolubles and preservation of the solubleactivity in aqueous environments, such stabilizers including certainpolyols, amino acids, polymers of amino acids having a charged sidegroup at physiological pH, and choline salts. Polyols are selected fromthe group consisting of non-reducing sugars, sugar alcohols, sugaracids, pentaerythritol, lactose, water-soluble dextrans and Ficoll;amino acids are selected from the group consisting of glycine,sarcosine, lysine or salts thereof, serine, arginine or salts thereof,betaine, N,N,-dimethyl-glycine, aspartic acid or salts thereof, glutamicacid or salts thereof; a polymer of an amino acid having a charged sidegroup at physiological pH may be selected from polylysine, polyasparticacid, polyglutamic acid, polyarginine, polyhistidine, polyornithine andsalts thereof; and choline derivatives are selected from the groupconsisting of choline chloride, choline dihydrogen citrate, cholinebitartrate, choline bicarbonate, tricholine citrate, choline ascorbate,choline borate, choline gluconate, choline phosphate,di(choline)sulphate and dicholine mucate.

U.S. Pat. No. 4,917,685 discloses a delivery device designed to beimplanted comprising growth hormone stabilized using the samestabilizers as mentioned in EP 303746.

Published European patent application No. 374,120 discloses a stabilizedformulation comprising hGH and a polyol having three hydroxy groups.Glycerol and tris(hydroxymethyl)aminomethane are mentioned. Furthermore,the presence of histidine hydrochloride as a buffer together with thepolyol is disclosed.

International Patent Publication No. WO 93/12811 discloses stabilizedformulations of growth hormone in the form of a lyophilized powder or anaqueous solution-comprising asparagine.

International Patent Publication No. WO 93/12812 discloses stabilizedformulations of growth hormone in the form of a lyophilized powder or anaqueous solution-comprising histidine. In such formulations thedeamidation is reduced by 25-30% as compared to a correspondingformulation of growth hormone comprising phosphate buffer.

International Patent Publication No. WO 93/19776 discloses proteinformulations comprising growth hormone comprising citrate as buffersubstance being more stable than formulations comprising phosphatebuffer. The formulations may also comprise amino acids such as glycineand alanine and/or mannitol or other sugar alcohols and/or glyceroland/or other carbohydrates and optionally a preservative such as benzylalcohol.

International Patent Publication No. WO 94/03198 discloses a stableaqueous formulation containing human growth hormone, a buffer, anon-ionic surfactant, and, optionally, a neutral salt, mannitol, or, apreservative.

SUMMARY OF THE INVENTION

It has now surprisingly been found that a formulation of human growthhormone comprising leucine as additive shows a very high stabilityagainst deamidation and oxidation. The stability of the product allowsfor the storing and shipment thereof in a lyophilized state or in theform of a dissolved or re-dissolved formulation. The invention isrelated to such a formulation. Additionally, the invention is related toa method for making such a formulation comprising adding the growthhormone to a solution comprising leucine. The invention is also directedto a method for treating a disorder associated with growth hormonedeficiency, comprising administering the pharmaceutical formulation ofclaim 7 in an amount effective to treat said deficiency.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical formulations of the invention may be formulated foradministration in any suitable way, e.g. by parenteral or oraladministration or administration to a mucosal membrane, e.g. nasaladministration. The pharmaceutical formulation may be presented in theform of a dose in a vial or cartridge or any other suitable containersuch as a prefilled syringe or a pen device.

Thus, the formulation of the invention may be in the form of alyophilized powder to be reconstituted later using conventional vehiclessuch as distilled water or water for injection or in the form of asolution comprising growth hormone. Such vehicles may compriseconventional preservatives such as m-cresol and benzyl alcohol.

A preferred embodiment of the invention is in the form of apharmaceutical formulation of human growth hormone comprising leucineand further comprising a carrier in the form of a buffered aqueoussolution of growth hormone. Such a formulation is in a ready-to-use formand may be stored and shipped as an aqueous solution without anyconsiderable degradation.

The pharmaceutical formulation of the invention may furthermore comprisesalts for adjusting the tonicity and/or an excipient in order tofacilitate the processing thereof, e.g. lyophilization and the rapid andcomplete dissolution of a lyophilized formulation when reconstitutingthe formulation before use. An excipient may be selected fromdisaccharides such as lactose, trehalose, and sucrose, sugar alcoholssuch as sorbitol or mannitol, polysaccharides such as the polymerscommercialized as Dextran® products such as Dextran® 40, Dextran® 70 orDextran® 75, and Ficoll® and polyvalent alcohols such as polyethyleneglycol or polyvinyl alcohol or a combination of two or more of these.

The pharmaceutical formulation may be prepared by adding growth hormoneto a solution comprising leucine, preferably in an amount of up to 100mM, more preferred in an amount of about 1-10 mM, preferably 2-6 mM,most preferred about 3-5 mM. The growth hormone may be in solid form ormay be in a buffer solution, e.g. histidine, citrate, tartrate orphosphate buffer. The leucine solution may be obtained by dissolvingleucine in deionized water optionally containing of benzyl alcohol. ThepH of said formulation may be adjusted from about 2 to about 8 morepreferred to pH from 5 to 7, especially to about 6.8 by adding an acidwhich has no adverse effect on the growth hormone, preferably aphysiologically acceptable acid e.g. a mineral acid such as hydrochloricacid, sulphuric acid or nitric acid or an organic acid such as aceticacid. In one embodiment of the method of the invention, salts and/or anexcipient may be added. In yet another embodiment, the solution isfilled into a container and lyophilized.

Still another aspect of the invention relates to the use of leucine forthe formulation of a stabilized formulation of growth hormone.

In the present context "growth hormone" may be growth hormone from anyorigin such as arian, bovine, equine, human, bovine, porcine, salmon,trout or tuna growth hormone, preferably bovine, human or porcine growthhormone, human growth hormone being most preferred. The growth hormoneused in accordance with the invention may be native growth hormoneisolated from a natural source, e.g. by extracting pituitary glands in aconventional manner, or a growth hormone produced by recombinanttechniques, e.g. as described in E. B. Jensen and S. Carlsen in Biotechand Bioeng. 36, 1-11 (1990). The "growth hormone" may also be amedicated form of growth hormone wherein one or more amino acid residueshas (have) been deleted; an analogue thereof wherein one or more aminoacid residues in the native molecule has (have) been substituted byanother amino acid residue, preferably a natural amino acid residue, aslong as the substitution does not have any adverse effect such asantigenicity or reduced action; or a derivative thereof, e.g. having anN- or C-terminal extension such as Met-hGH. The preferred growth hormoneis hGH.

The term "dose" of growth hormone refers to that amount that providestherapeutic effect in an administration regimen. The formulations hereofare prepared containing amounts of hGH at least about 0.1 mg/ml,preferably upwards of about 10 mg/ml, preferably from about 1 mg/ml toabout 40 mg/ml, more preferably from about 1 mg/ml to about 25 mg/ml,e.g. from 1 mg/ml to about 5 mg/ml, calculated on the ready-to-useformulation. For use of these compositions in administration to humanbeings suffering from hypopituitary dwarfism, for example, theseformulations contain from about 0.1 mg/ml to about 10 mg/ml,corresponding to the currently contemplated dosage regimen for theintended treatment. The concentration range is not critical to theinvention and may be varied by the physician supervising theadministration.

Leucine to be used in accordance with the present invention ispreferably naturally occurring alpha leucine. The amino acid(s) may be lor d amino acid(s) or a mixture thereof.

In the present context "high stability" is obtained when the formulationis more stable than the conventional formulation comprising phosphatebuffer and preferably as stable as a corresponding formulationcomprising histidine as stabilizer in which the de-amidation of hGH isreduced by approximately 20% as compared with phosphate buffer asdisclosed in WO 93/12812.

The solvent used in the formulations of the invention may be water,alcohols such as ethyl, n-propyl or isopropyl, butyl alcohol or mixturesthereof. The solvent may comprise a preservative such as m-cresol orbenzyl alcohol.

EXAMPLE

Reduction of the deamidation.

The rate of deamidation was examined at 37° C. for hGH formulationscomprising 4 mg/ml hGH at pH 6.8 in the presence of 5 mM L-leucine orD-leucine as compared to histidine at pH 6.8.

The hGH formulations were prepared by dissolving 8 mg hGH in 2 ml of 10mM solution of the amino acid. Thus, 2 ml 3.0% of benzyl alcohol wasadded to give a final formulation of 4 mg/ml hGH, 5 mM D- or L-leucineor histidine, 1.5% benzyl alcohol, pH 6.8 (adjusted adding HCl or NaOH).

The hGH formulations stated in the below table were stored at 37° C. for7 days and analyzed for the content of deamidated hGH by IE-HPLC. Theresults appear from the below table.

                  TABLE                                                           ______________________________________                                                            Corrected content                                                                         Contents of                                           pH start/pH of deamidated                                                                             desamido hGH as                               Formulation                                                                           end/% desamido                                                                            hGH*        compared with His                             ______________________________________                                        L-leucine                                                                             6.7/6.6/15.3                                                                              16.8        97                                            D-leucine                                                                             6.7/6.7/16.6                                                                              17.6        102                                           Histidine                                                                             6.7/6.8/16.8                                                                              17.3        100                                           ______________________________________                                         *Desamido corrected by 1% per 0.1 pH unit deviation from 6.8             

The contents of deamidated hGH in the starting material was: 2.0%

From the above table it appears that the deamidation of hGH is reducedby 25-30% by addition of D-leucine or L-leucine as compared withphosphate buffer (at least as stable as compared to histidine, cf. WO93/12812 above).

The above results show that the rate of deamidation is reduced insolutions containing L-leucine or D-leucine in a low concentration of upto 100 mM, preferably about 5 mM. The rate of deamidation may be reducedby more than 30% by substituting the phosphate buffer with L-leucine orD-leucine.

The use of benzyl alcohol as preservative seems to have no influence onthe rate of deamidation.

What is claimed is:
 1. A pharmaceutical formulation comprising a growthhormone and leucine.
 2. The pharmaceutical formulation according toclaim 1 in which said formulation is in the form of a buffered aqueoussolution.
 3. The pharmaceutical formulation according to claim 1 inwhich the formulation has a pH from about 2 to about
 8. 4. Thepharmaceutical formulation according to claim 1 wherein theconcentration of leucine is up to about 100 mM.
 5. The pharmaceuticalformulation according to claim 1 further comprising salts andsaccharides.
 6. The pharmaceutical formulation according to claim 1wherein the growth hormone is hGH.
 7. The pharmaceutical formulationaccording to claim 1 in which said formulation is in lyophilized form.8. A method of preparing the pharmaceutical formulation of claim 1comprising adding the growth hormone to a solution comprising leucine.9. The method according to claim 8 in which the solution comprisingleucine is obtained by dissolving leucine in deionized water.
 10. Themethod according to claim 8 in which the solution comprising leucinealso comprises benzyl alcohol.
 11. The method according to claim 8 inwhich further comprises adjusting the pH of said formulation to fromabout 2 to about
 8. 12. The method according to claim 8 which furthercomprises adding salts.
 13. The method according to claim 8 whichfurther comprises adding an excipient.
 14. The method according to claim8 which further comprises filling a solution comprising growth hormoneand leucine into a container and lyophilizing said solution.
 15. Amethod for treating a disorder in a patient affectable by growth hormonecomprising administering to the patient an amount of the pharmaceuticalformulation of claim 1 effective to treat said disorder.